The overall goal of the proposed work is to determine whether developmental or adult exposures to inorganic arsenic (iAs) increase prostate cancer (PCa) risk by reprogramming human prostate stem cells. Prior studies have revealed that environmental iAs exposure from natural sources (e.g. drinking water) increases PCa incidence. Recent findings also indicate that iAs directly alters stem cells and augments transformation and studies with PCa cell lines support that this may occur in the prostate gland. To further delineate iAs action(s) in normal human prostate stem cells, determine windows of sensitivity for human exposures and link iAs stem cell exposures to PCa development, novel human prostate stem cell models have been established using primary epithelial cells from disease-free human prostates and human embryonic stem cells (hESC). Importantly, in vivo models using either hESC or adult prostate stem cells mixed with rat inductive mesenchyme have been produced to generate chimeric prostate tissues with normal human prostate epithelium. These original approaches are in place to directly address several unresolved issues regarding iAs exposures and stem cell reprogramming events and to potentially link iAs to growth of human PCa. To accomplish this three specific aims are proposed. Specific Aim 1: Delineate the effects of iAs in modulating normal human prostate and embryonic stem cell self-renewal and differentiation to epithelial cell lineages. To accomplish this, FACS and prostasphere assay of adult human prostate stem cells and directed differentiation of hESC in vitro will be utilized to interrogate te actions of a range of iAs doses in perturbing normal prostate development and growth. Specific Aim 2: Elucidate the molecular underpinnings of As reprogramming of prostate stem cells by identifying DNA methylation modifications and resultant changes in gene expression. In parallel with Specific Aim 1, analysis of prostasphere DNA methylome, DNA hydroxymethylome and transcriptome will be undertaken to identify genome-wide marks in stem cells with resultant As gene expression signatures. Specific Aim 3: Determine whether As exposure initiates carcinogenesis, acts as a co-carcinogen or promotes PCa progression in human prostate epithelium using novel in vivo chimeric prostate models. Adult prostate stem cells from normal men, PCa patients or hESC will be used to generate chimeric tissues grown as renal grafts in nude mice. Carcinogenesis in normal epithelium will be monitored after iAs exposures alone or with steroids as co-carcinogens. PCa progression will be examined in PCa stem cell-derived grafts exposed to iAs. Finally, aberrant expression of iAs-reprogrammed genes will be evaluated in tissue microarrays constructed from PCa patients for translational relevance. The present approaches using fresh human specimens with in vivo models combined with state-of-the-art epigenomic technologies are a marked advance over current approaches that will undoubtedly provide significant new and useful information pertaining to human prostate health. Together, these studies will generate novel information on how iAs increases PCa risk and provide a rationale framework for iAs exposure assessment.